Methods and formulations for treating scars and beta-catenin-mediated disorders

ABSTRACT

A topical drug composition for treating a β-catenin-mediated disorder in a mammal is provided comprising a Nefopam compound selected from Nefopam, or a functionally equivalent analogue, prodrug, salt or solvate thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/516,016 filed on Dec. 15, 2010 which claims priority to U.S.Provisional Application Ser. No. 61/286,633, filed Dec. 15, 2009, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the treatment of scars andβ-catenin-mediated disorders.

BACKGROUND OF THE INVENTION

Fibroproliferative processes are a group of disorders characterized byan excessive proliferation of mesenchymal fibroblast-like spindle cells.They range from hypertrophic wounds to the development of neoplasms suchas aggressive fibromatosis (AF).

During wound healing, several cell types and signaling pathways areactivated to reconstitute the epithelial and dermis layers of the skin.Following cutaneous injury, three sequentially distinct but overlappingprocesses are initiated: inflammatory, proliferation, and remodeling.During the proliferative phase, mesenchymal fibroblast-like cellsaccumulate in the dermal component of the skin while the epithelial cellbarrier layer is reformed (Singer 1999, Martin 1997, McClain 1996).β-catenin has been shown to mediate epithelial and mesenchymal cellactivity, whereby it is able to increase proliferation anddifferentiation in dermal mesenchymal cells and decrease migration inepithelial keratinocytes (Cheon 2002). Mouse models have demonstratedthat β-catenin can modulate the resulting wound size, where inducedlevels of β-catenin by lithium treatment result in wound healing with alarger size (Cheon 2006). Also, a transgenic mouse in which stabilizedβ-catenin is expressed in mesenchymal cells, has been generated, undercontrol of a tetracycline-regulated promoter. Wounded mice healed withhyperplastic cutaneous wounds compared to wildtype control mice (Cheon2002). This demonstrates the importance of β-catenin in mesenchymalcells and its crucial role in wound healing.

Another fibroproliferative disorder mediated by β-catenin is aggressivefibromatosis (AF), also called desmoid tumour. AF is a locally invasivesoft tissue tumour comprised of mesenchymal fibroblast-like spindlecells. AF occurs as either a sporadic lesion or a familial syndrome,such as familial adenomatous polyposis (FAP). B-catenin stabilization isa universal occurrence in AF, as demonstrated by elevated β-cateninlevels and increase β-catenin-mediated transcriptional activity.Furthermore, β-catenin stabilization is sufficient to cause AF as shownusing a transgenic mouse model that over-expresses the stabilized formof β-catenin (Cheon 2002). This suggests a crucial role β-catenin playsin fibroproliferative disorders and its importance in mesenchymal cells.

In addition to a role for β-catenin in fibroproliferative disorders, anumber of studies have demonstrated deregulated β-catenin expression isan important event in the genesis of a number of malignancies, such ascolon cancer, melanoma, hepatocellular carcinoma, ovarian cancer,endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer.β-catenin mutations appear to be a crucial step in the progression of asubset of these cancers, suggesting an important role in the control ofcellular proliferation or cell death (as described in Polakis P. Themany ways of Wnt in cancer. Curr Opin Genet Dev. 2007 February;17(1):45-51).

In view of foregoing, it is desirable to develop novel methods effectiveto treat conditions and disorders that may be associated with β-catenin.

SUMMARY OF THE INVENTION

It has now been found that Nefopam, and analogues thereof, are useful totreat disorders mediated by β-catenin, such as fibroproliferativedisorders, as well as treating scar tissue.

Accordingly, in one aspect of the invention, a topical formulation ofNefopam is encompassed, comprising a compound selected from Nefopam or afunctionally equivalent salt or solvate thereof; at least one fattyalcohol emulsifier selected from the group consisting of cetyl alcoholand stearyl alcohol; an oil-in water emulsifier; at least onepreservative; at least one penetration enhancer selected from the groupconsisting of diethylene glycol monoethyl and isopropyl myristate; andsodium hydroxide.

In another aspect, the invention encompasses methods of reducing thesize of a dermal scar comprising providing a topical drug formulationtwice per day beginning on about day 5 to about day 7 followingwounding, wherein the formulation comprises one percent Nefopam HCl,emulsifiers, at least one penetration enhancer, and at least onepreservative, and further comprises a pH of about 5.3.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in further detail withreference to the following Figures:

FIG. 1A is a bar graph indicating cell viability in cultured normalfibroblast cells and cultured cells derived from two hyperplastic woundsfollowing treatment with DMSO (control) or Nefopam using the SRB assay.Percent cell survival is given as a mean and 95% confidence interval.There is significant decline in the percent of cells surviving incultures treated with Nefopam compared to DMSO control hyperplasticwound cell cultures, however, cell survival rates in normal fibroblastcultures remained relatively unchanged (asterisk indicates significancecompared to normal fibroblast cultures).

FIG. 1B is a western blot analysis of β-catenin levels in hyperplasticwound cell cultures. Nefopam treatment was shown to substantially reduceβ-catenin protein levels compared to DMSO treated controls.

FIG. 2 is a graph comparing the number of aggressive fibromatosis (AF)tumours formed in male Apc+/Apc1638N mice left untreated or treated withNefopam or DMSO control and illustrating the number ofepithelial-derived polyps in the upper gastrointestinal tract under thesame treatment. 1) No Treatment (n=11), 2) 0.1% DMSO (n=10), and 3)Nefopam at 40 mg/kg body weight (n=10).

FIG. 3A is a western blot of β-catenin protein levels (92 kDa) inextracts from primary cell cultures derived from human aggressivefibromatosis (AF) tumours (n=5) following treatment for 5 days with oneof 0.1% DMSO (control) or Nefopam. β-catenin protein levels were alsodetermined in primary fibroblast cell cultures incubated with Wnt3a withor without Nefopam. Experiments were performed in triplicate. Actinexpression is shown as a lysate loading control.

FIG. 3B is a graph of densitometry analysis of protein level datashowing a nearly 5-fold decrease in total β-catenin protein levels incell cultures derived from human AF tumours treated with 0.1% DMSO(control) or Nefopam. Means and 95% confidence intervals are shown.Statistically significant differences (p<0.05) compared to the controlare indicated by an asterisk.

FIG. 4A is a graph showing the means and 95% confidence intervals ofcell viability of primary cells derived from human AF tumours treatedwith DMSO (n=5) or Nefopam (n=5) for 5 days. Cell viability was measuredby staining cells with Trypan Blue Dye and counting both live (clear)and dead (blue) cells. Nefopam significantly decreased the number oflive cells while the number of dead cells did not change. Statisticallysignificant differences (p<0.05) compared to controls are indicated withan asterisk

FIG. 4B is a graph showing percent BrdU-positive/DAPI-positive cellscompared to total DAPI-positive cells as a measure of proliferation incultures of primary cells derived from human aggressive fibromatosistumours (n=2) treated with DMSO or Nefopam in triplicate for 5 days.Nefopam significantly reduces the incorporation of BrdU into cells. Themeans and 95% confidence intervals are shown. Statistically significantdifferences (p<0.05) compared to the control are indicated by asterisk.

FIG. 5A shows western blot analysis of lysates extracted fromimmortalized human fibroblast cells. A significant decrease in totalβ-catenin protein levels in cells treated with Nefopam compared to cellstreated with DMSO was observed. GAPDH expression is shown as a lysateloading control.

FIG. 5B is a graph of densitometry data corresponding to western blotdata of FIG. 5A.

FIG. 6A is a western blot analysis of β-catenin protein levels in cellcultures from Tcf mice wounds 14 days post-wounding. GAPDH expression isshown as lysate loading control.

FIG. 6B is a graph of normal scar size in mice subjected to fullthickness circular wounds following treatment with either Nefopamformulated with a carrier (Nefopam) or carrier alone (control)administered systemically as 40 mg/kg daily for two weeks. The graphshows the mean and 95% confidence interval for the diameter of thesurface area of a cutaneous wound generated using a 4 mm biopsy punch.The diameter of the wound is significantly smaller following Nefopamtreatment compared to control treatment (asterisk indicates asignificant difference).

FIG. 7 is a line graph indicating relative β-catenin protein levels overtime (measured in weeks) during normal wound healing (normal) and inhyperplastic wounds (hyperplastic) compared to unwounded tissue. Thenormal pattern of rise and fall of β-catenin protein levels duringnormal wound healing is deregulated in hyperplastic wounds, whichexhibit a significantly prolonged duration of elevated β-catenin proteinlevels.

FIG. 8 is a graph of the mean and 95% confidence interval for thediameter of the surface area of cutaneous hyperplastic scars four weekspost-wounding. 4 mm diameter full thickness circular wounds weregenerated using a biopsy punch. Wound diameter is given in mm. Anasterisk indicates statistically significant differences in scar sizenoted when compared to treatment with TGF-β (p<0.01), where TGF-βinjection at the time of wounding is known to cause hyperplastic scarsof increased size.

FIG. 9 is a graph of varying concentration Nefopam topical formulationsin three different carriers: carboxymethylcellulose (CMC), petrolatum,and hypromellose. The three carriers were tested in vivo in a mousemodel to determine the formulation most effective in delivering Nefopamthrough the skin. Petrolatum-based carrier formulations demonstratedenhanced Nefopam release properties as determined by measurement ofNefopam levels in the skin and serum.

FIG. 10 is a graph of relative scar surface area measured in arbitraryunits (the scar size upon wounding is considered as 100 arbitraryunits). Full thickness puncture wounds 4 mm in diameter were treatedtopically with either carrier control cream or 1% Nefopam creamformulated in petrolatum carrier twice daily for 14 days. The datarepresent an average of 10 wounds per treatment with standard deviation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method of treating a β-catenin-mediated disorder in a mammal isprovided comprising administering Nefopam or a functionally equivalentanalogue thereof to the mammal.

As used herein, the term “β-catenin-mediated disorder or condition”refers to disorders or conditions characterized by the accumulation offibrous tissue (“fibrosis”) including, but not limited to,fibroproliferative disorders such as dermal scars includinghypertrophic, hyperplastic and keloid scars, either in formation oralready formed, and aggressive fibromatoses e.g. sporadic lesion or afamilial syndrome such as familial adenomatous polyposis (FAP), liverfibrosis, lung fibrosis (e.g., silicosis, asbestosis), kidney fibrosis(including diabetic nephropathy), glomerulosclerosis, Lederhose diseaseand Dupuytren's contracture (DC), as well as malignancies, such as coloncancer, colorectal cancer, melanoma, hepatocellular carcinoma, ovariancancer, endometrial cancer, medulloblastoma pilomatricomas, and prostatecancer.

The term “Nefopam” refers to5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine andpharmaceutically acceptable functionally equivalent analogues, prodrugs,salts and solvates thereof. The term “functionally equivalent”, as itused with respect to analogues, prodrugs, salts and solvates of Nefopam,refers to the ability of the selected compound to modulate β-catenin.The extent to which the selected compound may modulate β-catenin mayvary from compound to compound.

The term “analogue” as used herein refers to compounds having thefollowing general formula (1),

wherein R₁ is H, C₁-C₆ alkyl optionally substituted with F orC₃-C₆cycloalkyl or C₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2;one of W, X, Y and Z is N, CH or CR₃ and the others are CH; R₂ is C₅-C₆heteroaryl, C₅-C₁0 cycloalkyl or cycloalkenyl optionally containing oneor more heteroatoms selected from O, N and S(O)_(n) where n is 0-2, andoptionally substituted with R₃; or a phenyl group optionally substitutedin one or more positions with one or more substituents independentlyselected from halogen, CN, CF₃, C₁-C₆ alkyl and OR₁, or the phenyl groupis fused to a five or six membered ring which may be carbocyclic,heterocyclic (containing 1-2 heteroatoms selected from O, N and S),aromatic or heteroaromatic (containing 1-2 heteroatoms selected from Oand N); R₃ is selected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂; COR₅;CO₂R₅; CON(R₅)₂; NR₁COR₄; NR₁SO₂R₄; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆ alkylsubstituted with R₃; C₁-C₆ alkyl optionally substituted withunsubstituted R₃; C₃-C₆cycloalkyl optionally substituted withunsubstituted R₃; C₂-C₆alkenyl optionally substituted with unsubstitutedR₃; C₂-C₆alkynyl optionally substituted with unsubstituted R₃; aryloptionally substituted with unsubstituted R₃; and five or six memberedaromatic heterocycles containing 1-4 heteroatoms selected from N and O;R₄ is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; wherein R₁ isH, C₁-C₆ alkyl, optionally substituted with F or C₃-C₆cycloalkyl orC₂-C₆ alkenyl; R₂ and R₃ are the same or different and are H, a halogen,CN, CF₃, C₁-C₆ alkyl or OR₁, or R₂ and R₃ form a five or six memberedring which may be carbocyclic, heterocyclic (containing 1-2 heteroatomstaken from O, N and S), aromatic or heteroaromatic (containing 1-2heteroatoms taken from O and N); one of W, X, Y and Z is N, or CR₄ andthe others are each CH; R₄ is a halogen atom, CF₃, CN, OR₇, SO₂N(R₆)₂,COR_(E), CO₂R₆, CON(R₆)₂, NR₁COR₅, NR₁SO₂R₅, NR₁CO₂R₅, NR₁CON(R₆)₂,OC₁-C₆ alkyl optionally substituted with R₄, C₁-C₆ alkyl optionallysubstituted with R₄, C3-C₆cycloalkyl optionally substituted with R₄,C₂-C₆alkenyl optionally substituted with R₄, C₂-C₆alkynyl optionallysubstituted with R₄, aryl optionally substituted with R₄, or a five orsix membered aromatic heterocycle containing 1-4 heteroatoms selectedfrom N and O, linked either through carbon or nitrogen; R₅ is C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl;each R₆ (which may be the same or different) is H, C₁-C₆ alkyl,C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl; and R₇is aryl or heteroaryl; or a pharmaceutically acceptable salt thereof;wherein R₁ is H, C₁-C₆ alkyl optionally substituted with F orC₃-C₆cycloalkyl or C₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2;one of W, X, Y and Z is N, CH or CR₃ and the others are CH; R₂ isC₅-C₆heteroaryl, C₅-C₁₀ cycloalkyl or cycloalkenyl optionally containingone or more heteroatoms selected from O, N and S(O)_(n) where n is 0-2,and optionally substituted with R₃; or a phenyl group optionallysubstituted in one or more positions with one or more substituentsindependently selected from halogen, CN, CF₃, C₁-C₆ alkyl and OR₁, orthe phenyl group is fused to a five or six membered ring which may becarbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, Nand S), aromatic or heteroaromatic (containing 1-2 heteroatoms selectedfrom O and N); R₃ is selected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂;COR₅; CO₂R₅; CON(R₅)₂; NR₁COR₄; NR₁SO₂R₄; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆alkyl substituted with R₃; C₁-C₆ alkyl optionally substituted withunsubstituted R₃; C₃-C₆cycloalkyl optionally substituted withunsubstituted R₃; C₂-C₆alkenyl optionally substituted with unsubstitutedR₃; C₂-C₆alkynyl optionally substituted with unsubstituted R₃; aryloptionally substituted with unsubstituted R₃; and five or six memberedaromatic heterocycles containing 1-4 heteroatoms selected from N and O;R₄ is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; wherein R₁ isH, C₁-C₆ alkyl optionally substituted with F or C₃-C₆cycloalkyl orC₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2; one of W, X, Y andZ is N, CH or CR₃ and the others are CH; R₂ is C₅-C₆ heteroaryl, C₅-C₁0cycloalkyl or cycloalkenyl optionally containing one or more heteroatomsselected from O, N and S(O)_(n) where n is 0-2, and optionallysubstituted with R₃; or a phenyl group optionally substituted in one ormore positions with one or more substituents independently selected fromhalogen, CN, CF₃, C₁-C₆ alkyl and OR₁, or the phenyl group is fused to afive or six membered ring which may be carbocyclic, heterocyclic(containing 1-2 heteroatoms selected from O, N and S), aromatic orheteroaromatic (containing 1-2 heteroatoms selected from O and N); R₃ isselected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂; COR₅; CO₂R₅; CON(R₅)₂;NR₁COR₄; NR₁SO₂; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆ alkyl substituted withR₃; C₁-C₆ alkyl optionally substituted with unsubstituted R₃;C₃-C₆cycloalkyl optionally substituted with unsubstituted R₃;C₂-C₆alkenyl optionally substituted with unsubstituted R₃; C₂-C₆alkynyloptionally substituted with unsubstituted R₃; aryl optionallysubstituted with unsubstituted R₃; and five or six membered aromaticheterocycles containing 1-4 heteroatoms selected from N and O; R₄ isC₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; or wherein:R₁ is H, C₁-C₆ alkyl, optionally substituted with F or C₃-C₆ cycloalkylor C₂-C₄ alkenyl; R₂ and R₃ are the same or different and are each H,halogen, CN, CF₃, C₁-C₆ alkyl or OR₁, or R₂ and R₃ may form a five orsix membered ring which may be carbocyclic, heterocyclic (containing 1-2heteroatoms taken from O, N and S), aromatic or heteroaromatic(containing 1-2 heteroatoms taken from O and N); and one of W, X, Y andZ is N, CH or CR₄ and the others are CH; R₄ is halogen; CF₃; CN; OR₇;SO₂N(R₆)₂ (where each R₆ is the same or different); COR_(E); CO₂R₆;CON(R₆)₂ (where R₆ is the same or different); NR₁COR₅; NR₁SO₂R₅;NR₁CO₂R₅; NR₁CON(R₆)₂ (where each R₆ is the same or different), OC₁-C₆alkyl substituted with unsubstituted R₄, C₁-C₆ alkyl optionallysubstituted with unsubstituted R₄, C₃-C₆cycloalkyl optionallysubstituted with unsubstituted R₄, C₂-C₆alkenyl optionally substitutedwith unsubstituted R₄, C₂-C₆alkynyl optionally substituted withunsubstituted R₄ and aryl optionally substituted with unsubstituted R₄,or R₄ is a five or six membered aromatic heterocycle containing 1-4heteroatoms taken from N and O; R₅ is C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl; R₆ can be H,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₇ is aryl or heteroaryl; or a pharmaceuticallyacceptable salt thereof. Additional analogues of Nefopam are describedin WO2004/056788, WO2005/103019 and US2006/0019940, the contents ofwhich are incorporated herein by reference. Nefopam and analoguesthereof may be made using chemical synthetic methods well-known to thosein the art. In addition, Nefopam is commercially available.

The term “prodrug” refers to a compound (e.g. a drug precursor) that istransformed in vivo to yield a compound having the structure of Nefopamor an pharmaceutically acceptable analogue, salt, hydrate or solvatethereof. The transformation may occur by various mechanisms (e.g., bymetabolic or chemical processes), such as, for example, throughhydrolysis in blood. The term “salt(s)”, as employed herein, denotesacidic salts formed with inorganic and/or organic acids, as well asbasic salts formed with inorganic and/or organic bases. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful. A “solvate” is formedby admixture of Nefopam or an analogue thereof in a solvent which ispreferably pharmaceutically acceptable.

The present method encompasses the treatment of a β-catenin-mediateddisorder in a mammal. The terms “treat”, “treating” and “treatment” areused broadly herein to denote methods that favorably alter the targeteddisorder, including those that moderate or reverse the progression of,reduce the severity of, or prevent, the disorder. The term “mammal” isused herein to encompass both human and non-human mammals.

A method of treating dermal scars, including scars resulting from cuts,scrapes, infection, acne, burns, surgery, etc., hypertrophic,hyperplastic, keloid, scars involving mesenchymal andmesenchymal-derived cells, any of which may be β-catenin-mediated ornot, is also provided. The method comprises administering to the targetsite a therapeutically effective amount of a Nefopam compound. Themethod of treating scars, in formation or already formed, includesreducing the size of the scar (for example, by at least about 5-10%,preferably by at least about 20%, and more preferably by at least about25% or more) or prevalence of the scar (e.g. elevation of the scar,redness, etc.) and thereby improving the appearance thereof. In thisregard, as one of skill will appreciate, a scar assessment scale, e.g.the Manchester Scale, may be used to assess the improvement of a givenscar. The Manchester Scale assesses colour compared with surroundingskin, matte or shiny appearance, contour (flush with surrounding skin toscar/keloid), texture (normal to hard), margins (distinct or not), sizeand number (single or multiple) (Disability & Rehabilitation, 2009, Vol.31, No. 25: Pages 2055-2063; International Journal of Lower ExtremityWounds December 2007 6: 249-253).

Thus, Nefopam compounds may be utilized in a cosmetic treatment toreduce scar tissue and improve the aesthetics of the scar andsurrounding area, and may provide additional cosmetic features, e.g.anti-wrinkling effects.

In another embodiment, a method of treating tumours is provided. Tumourtreatment includes inhibiting tumour initiation and tumour cellproliferation. The method is useful to treat tumours resulting fromderegulated β-catenin expression such as aggressive fibromatosis, aswell as tumours resulting from various cancers such as colon cancer,melanoma, hepatocellular cancer, ovarian cancer, endometrial cancer andprostate cancer. The method comprises administering to a mammal in needof treatment, i.e. a mammal having a tumour, an effective amount ofNefopam, an analogue thereof, or a pharmaceutically acceptable salt,solvate or prodrug thereof.

While not wishing to be bound by any particular theory, treatment inaccordance with the present invention may be effected by the regulationor modulation of β-catenin expression, at the nucleic acid level, or theregulation or modulation of β-catenin activity, at the protein level.

Therapeutically effective dosages of Nefopam are administered to amammal in accordance with the invention. The term “therapeuticallyeffective” as it is used herein with respect to dosages refers to adosage that is effective to treat a β-catenin-mediated disorder withoutcausing unacceptable adverse side effects. The term “administered”refers to any appropriate means of providing Nefopam to a mammal, andwill depend on the dosage form being used as will be described. Forexample, the dosage may be administered orally, by injection, mucosallyand topically as will be described in more detail.

Therapeutically effective dosages according to the method, thus, are inthe range of about 0.0001 to about 1500 mg, for example, in a range ofabout 0.0001-100 mg. However, as one of skill in the art willappreciate, the effective therapeutic dosage of Nefopam, or analoguesthereof, will vary depending many factors, including but not limited to,the type of disorder to be treated, the nature and severity of thedisorder, the mammal to be treated, the symptoms of the mammal beingtreated, the compound used for the treatment, and the route ofadministration.

Nefopam may be administered in accordance with methods of the inventionalone or in a composition combined with a pharmaceutically acceptableadjuvant or carrier. The expression “pharmaceutically acceptable” meansacceptable for use in the pharmaceutical arts, i.e. not beingunacceptably toxic, or otherwise unsuitable for administration to amammal. Examples of pharmaceutically acceptable adjuvants include, butare not limited to, diluents, excipients and the like. Reference may bemade to “Remington's: The Science and Practice of Pharmacy”, 21st Ed.,Lippincott Williams & Wilkins, 2005, for guidance on drug formulationsgenerally. The selection of adjuvant depends on the intended mode ofadministration of the composition. In one embodiment of the invention,the compounds are formulated for administration by infusion, or byinjection either subcutaneously or intravenously, and are accordinglyutilized as aqueous solutions in sterile and pyrogen-free form andoptionally buffered or made isotonic. Thus, the compounds may beadministered in distilled water or, more desirably, in saline,phosphate-buffered saline or 5% dextrose solution. Compositions for oraladministration via tablet, capsule, lozenge, solution or suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, an elixir or syrup are prepared using adjuvants includingsugars, such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and derivatives thereof, includingsodium carboxymethylcellulose, ethylcellulose and cellulose acetates;powdered tragancanth; malt; gelatin; talc; stearic acids; magnesiumstearate; calcium sulfate; vegetable oils, such as peanut oils, cottonseed oil, sesame oil, olive oil and corn oil; polyols such as propyleneglycol, glycerine, sorbital, mannitol and polyethylene glycol; agar;alginic acids; water; isotonic saline and phosphate buffer solutions.Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers,tableting agents, disintegrating agents, anti-oxidants, preservatives,colouring agents and flavouring agents may also be present. In anotherembodiment, the composition may be formulated for application topicallyas a cream, lotion or ointment. For such topical application, thecomposition may include an appropriate base such as a triglyceride base.Such creams, lotions and ointments may also contain a surface activeagent and other cosmetic additives such as skin softeners and the likeas well as fragrance. Aerosol formulations, for example, for nasaldelivery, may also be prepared in which suitable propellant adjuvantsare used. Compositions of the present invention may also be administeredas a bolus, electuary, or paste. Compositions for mucosal administrationare also encompassed, including oral, nasal, rectal or vaginaladministration for the treatment of infections which affect these areas.Such compositions generally include one or more suitable non-irritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax, a salicylatc or other suitablecarriers. Other adjuvants may also be added to the compositionregardless of how it is to be administered which, for example, may aidto extend the shelf-life thereof.

In accordance with the present method, a Nefopam compound may beadministered in a convenient manner by any of a number of routesincluding but not limited to oral, subcutaneous, intravenous,intraperitoneal, intranasal, enteral, topical, sublingual,intramuscular, intra-arterial, intramedullary, intrathecal, inhalation,ocular, transdermal, vaginal or rectal means. Nefopam compounds may alsobe administered to cells in ex vivo treatment protocols. Depending onthe route of administration, Nefopam compounds may be coated or encasedin a protective material to prevent degradation by, e.g. enzymes, acidsor other conditions that may affect the therapeutic activity thereof.

In one embodiment, Nefopam, or an analogue thereof, may be topicallyapplied to a target site, e.g. a scar in formation or already formed,affixed to a biocompatible device, polymer or other matrix, e.g. such asa bandage, dressing, polymer mesh, implant, device or other cosmeticallyrelated item. Dermal fibroblasts/keratinocytes bioengineered to expressa Nefopam compound may also be applied to a target site. A suitablematrix or polymer mesh, e.g. artificial or non-artificial skin grafts,may alternatively be impregnated with a Nefopam compound for applicationto a target site to permit slow-release of the compound for continuoustreatment of the site over a period of time.

Formulation of topical embodiments of the invention require the use ofsolvents, solubilizers, emulsifiers, thickening agents, dispersants,preservatives, buffers, chelating agents, and penetrating agents.

In certain topical embodiments of the invention Transcutol® P is used.It is a diethylene glycol monoethyl ether solvent and that is also as asolubilizer for poorly water-soluble active pharmaceutical ingredients.It is used to achieve improved drug penetration, permeation and a drugdepot effect.

In certain topical embodiments of the invention, fatty alcohols such ascetyl alcohol and stearyl alcohol, are used as an emulsifier andthickening agent.

Topical embodiments may also contain Liposorb® S-20 (Polysorbate 60 NF)as an oil-in-water emulsifier to produce a fine-textured cream.Liposorb® S-20 also adds watcr-dispersibility and is a solubilizer.

Propyl Paraben NF may be used in embodiments of the invention as apreservative. This is particularly important in topical embodiments ofthe invention to be applied by patients on an out-patient basis.

Lexol® IPM-NF is a high purity isopropyl myristate solvent for topicaluse. In certain embodiments of the invention it improves penetration.The low odor and light color makes it ideal for topical embodiments ofthe invention where high emollient concentrations are required. It has alow viscosity, low freezing point, and good spreading properties. Incertain embodiments Lexol® also enhances fluidity during cold storageand depress the cloud point of the formulation.

Methylparaben NF is a natural anti-fungal agent used in certain topicalembodiments of the invention. It is a fine white, powder, odorless andsoluble in water.

VERSEN NF (EDTA or ethylenediaminetetraacetic acid) is a chelating agentused in certain topical embodiments of the invention that provides metalion control and is used for stabilization, helping to maintain potencyof the Nefopam.

In some embodiments sodium hydroxide may be used for pH adjustment toachieve the desired pH of 5.0 to 5.6.

The present Nefopam compounds may be administered in a controlledrelease formulation using well-established methods including, forexample, by dissolution or diffusion-controlled monolithic devices,beaded encapsulated systems, osmotically controlled systems, andmodified film coating systems incorporating suitable polymeric andnon-polymeric hydrophilic and hydrophobic materials. Suitablecontrolled-release formulations may include hydrophilic materialscomprising, but not limited to, acrylic or methacrylic polymers orcopolymers, alkylvinyl polymers, celluloses, hydroxyalkyl celluloses,carboxyalkyl celluloses, polysaccharides, alginates, pectins, starchesand derivatives, natural and synthetic gums, polycarbophil, chitosans.Suitable hydrophobic materials comprise, but are not limited to,hydrophobic polymers, waxes, fats, long-chained fatty acids, theircorresponding esters, their corresponding ethers, and their mixtures.

In another embodiment, Nefopam compounds may be administered incombination with one or more additional therapeutic agents, includingfor example, an anti-scarring agent; a wound healing agent such as agrowth factor, e.g. epidermal growth factor, bFGF, PDGF; platelets,dermal fibroblasts and keratinocytes; chemotherapeutic agents such as,but not limited to, rapamycin, troglitazone, rosiglitazone, celecoxib,retinoids and iressa. In this regard, Nefopam may be administered in aseparate formulation, or together with an additional therapeutic agentin a combined formulation.

The stimulation of the calcium stimulating receptor, CaSR, by Ca²⁺produced a striking and time-dependent decrease in the phosphorylationthat promotes β-catenin transcriptional activity. The reducedphosphorylation of β-catenin coincides with a decline in its nuclearlocalization and a marked redistribution to the plasma membrane.Furthermore, CaSR stimulation by Ca²⁺ promoted a down-regulation ofβ-catenin-mediated transcriptional activation. Thus, in certainembodiments of the invention, Ca²⁺ may be added to the drug form toenhance the β-catenin inhibiting activity of Nefopam (The Journal ofBiological Chemistry, 287, 1158-1167 (2012)). Various forms of calciummay be employed including calcium gluconate NF.

In addition, the present methods may be utilized in a combination withother therapies, for example, in combination with radiation therapy inthe treatment of malignancies, or in combination with laser therapy totreat scar tissue such as normal scars, hyperplastic scar tissue and thelike.

In a further aspect of the invention, an article of manufacture isprovided comprising packaging and a composition comprising Nefopam asdescribed. The packaging is labelled to indicate that the composition issuitable to treat a β-catenin-mediated disorder, or may be labelled toindicate that the composition is suitable to treat scarring, either information or already formed.

The present invention is described by reference to the accompanyingFigures and specific examples which are not to be construed as limiting.

EXAMPLES

The following materials and methods were used in the examples discussedbelow.

Apc⁺/Apc^(1638N) AF Mouse Model and Treatment Plan.

The generation and phenotype of Apc/Apc1638N mice have been wellcharacterized. These mice harbour a targeted mutation at codon 1638 inthe Apc gene as a result of a neomycin insert in antisense orientationat exon 15. Male mice develop an average of 45 AF lesions and 6gastrointestinal polyps by the age of 6 months, while female micedevelop significantly fewer numbers of AF lesions. Male Apc/Apc1638Nmice were divided into three study groups: No Treatment (n=11), 0.1%DMSO (n=10), and Nefopam at 40 mg/kg body weight (n=10). Treatment bydaily oral gavaging began 2 months after Apc/Apc1638N mice were weanedand continued for 3 months. At autopsy, AF tumours and intestinal polypswere scored macroscopically. AF tumours and normal tissue were collectedfor protein extraction and fixed for histological examination.

Tcf Reporter Mice and Wounding Experiments.

A Tcf-reporter construct containing the lacZ gene downstream of a c-Fosminimal promoter and three consensus Tcf-binding motifs was constructed.Upon binding of β-catenin/Tcf complex to Tcf motifs, the expression oflacZ is activated. Tcf mice were wounded as described previously: two 4mm diameter full-thickness skin wounds were generated using a dermalbiopsy punch (Miltex Instrument Company, York, Pa., USA). Wounded Tcfmice were separated into two study groups: Control group, which receiveddaily intraperitoneal injections of Saline; and Nefopam group, whichreceived daily intraperitoneal injections of 40 mg/kg body weight. At 14days post-wounding, wound sizes were examined, and wound tissues werecollected for RNA and protein extraction and fixed for histologicalexamination.

Human AF Tumour and Normal Fascial Tissue Samples.

Samples of human aggressive fibromatosis tumours were obtained at thetime of surgery from the Hospital for Sick Children, Toronto. Tumourtissue and surrounding normal fascial tissue from the same patient wereharvested and processed immediately after surgical excision. Tissueswere cryopreserved and stored in liquid nitrogen vapour.

Cell Culture Studies.

Primary cell cultures from the human AF tumour and normal fascial tissuesamples were established. Monolayer cultures were cultured in DMEMsupplemented with 10% fetal bovine serum and maintained at 37° C. in 5%CO₂. Cells were divided when confluent and experiments were performedbetween the first and fifth passages. Prior to experimental studies,cells were seeded overnight and treatment began the following day (Day0) where cells were treated with vehicle control 0.1% DMSO with orwithout 250 μm Nefopam prepared in DMEM media.

Cell Viability Assay, Proliferation Assay and Apoptosis Assay werePerformed.

Cell viability was measured using the Trypan Blue Dye Exclusion method.Cells were stained with Trypan Blue Dye at a 1:1 ratio, and both live(clear) and dead (blue) cells were accounted for. Proliferation wasmeasured using 5-bromo-2-deoxy-uridine (BrdU) Incorporation assay. AfterBrdU incubation for 12 hours, cells with incorporated BrdU wereidentified using rabbit monoclonal anti-BrdU antibody and horseanti-mouse antibody conjugated to Alkaline Phosphatase. Presence of BrdUwas detected using Alkaline Phosphatase substrate. Percentage ofpositively stained nuclei out of total nuclei was analyzed over 10high-powered fields.

Protein Extraction and Western Blot Analysis.

Tissue samples were washed twice with PBS and lysed with Reporter GeneAssay Lysis Buffer (Roche). Lysates were centrifuged at 16,000×g for 5minutes to remove cell debris and quantified using the BicinchoninicAcid (BCA) Protein Assay (Pierce). Equal amounts of total protein wereseparated by electrophoresis through an SDS-polyacrylamide gel,transferred to a nitrocellulose membrane (Amersham), and immunoblottedovernight at 4° C. with primary antibodies against phosphoGSK3β (Ser 9,rabbit polyclonal, New England Biolabs), β-catenin (mouse monoclonal,Upstate Biotechnology), total GSK3β (mouse monoclonal, TransductionLaboratories), and GAPDH (mouse monoclonal, Upstate Biotechnology).Horseradish Peroxidase (HRP)-tagged secondary antibodies and EnhancedChemiLuminescence (Amersham) were used to detect hybridization.Densitometery was performed using the AlphaEaseFC software (AlphaInnotech). Western blotting was performed in triplicates to ensurereproducibility.

Statistical Analysis.

Data in this work are presented as mean±95% confidence intervals. Allstudies were performed in at least triplicates to ensurereproducibility.

Example 1: Nefopam Treatment Reduces Hyperplastic Wound Cell Viability

Compounds were screened to identify those that meet two criteria: 1)inhibit cell viability of fibroblasts obtained from hyperplastic woundswhich exhibit β-catenin activation; and 2) show little to no effect onnormal dermal fibroblast cultures. The biological relevance of thescreen was considerable since cells used for the screen were obtainedfrom patients with hyperplastic wounds as well as healthy tissue. Theexperiments were repeated in triplicate within 96 well plates, with eachwell containing 4000 cells treated with between 0.1 1.0, or 10 μM ofcompound or DMSO as a control. The Sulforhodamine B assay (SRB) was usedto measure cell viability. Compounds detected within the initial screenunderwent further testing using a larger pool of samples, from whichNefopam was identified (see FIG. 1A).

β-catenin levels in cell cultures from hyperplastic scars treated withNefopam or control were analyzed using Western blot analysis. It wasobserved that Nefopam substantially reduced the protein level ofβ-catenin in cell cultures from hyperplastic wounds (see FIG. 1B). GAPDHexpression was included as a loading control.

Example 2: Nefopam Decreases the Number of AF Tumours Formed inApc/Apc1638N Mice

It was investigated whether or not Nefopam treatment was able tomodulate the phenotype of AF lesions in vivo. The number of AF tumoursformed in male Apc/Apc1638N mice treated with Nefopam was significantlyreduced compared to the number formed in untreated mice or mice treatedwith 0.1% DMSO at 6 months of age (8.18±1.77 vs 13.2±2.30 or 12.09±1.31,p<0.03, see FIG. 2). There were no significant differences in the numberof epithelial-derived polyps in the upper gastrointestinal tract (seeFIG. 2). This shows that Nefopam inhibits tumour initiation and furtheris specific to mesenchymal cells.

Example 3: Nefopam Decreases β-Catenin Levels in Human AF Tumour Cells

AF tumours are characterized by an increase in β-catenin levels. Toexamine whether Nefopam has the capacity to modulate β-catenin levels,primary cell cultures derived from several human AF tumours werestudied. Western blot analysis using an antibody against total β-catenindemonstrated a marked decrease in the amount of protein at size 92 kDaconsistent with total β-catenin as a result of Nefopam treatment for 5days, see FIG. 3A. Densitometry analysis showed nearly a 5-fold decreasein total β-catenin levels in human AF tumour cell cultures treated withNefopam compared to those treated with 0.1% DMSO (see FIG. 3B). Actinexpression was determined as a lysate loading control.

Example 4: Nefopam Decreases Cell Viability and Cell Proliferation inHuman AF Tumour Cells

To determine how Nefopam may modify AF cell behaviour, primary cellcultures derived from several human AF tumours were studied. First, theeffects of Nefopam on cell viability in human AF tumours were studied. Asignificantly smaller number of live cells were observed in human AFtumour cell cultures following Nefopam treatment compared to culturestreated with 0.1% DMSO (p<0.05). There were no significant differencesin the number of dead cells counted as a result of Nefopam treatment forthe tumours (p<0.05) (see FIG. 4A).

Upon demonstrating that β-catenin levels are involved in the regulationof the rate of proliferation in mesenchymal cells, the effects ofNefopam on proliferation in primary cell cultures were investigated.Using the BrdU incorporation assay, the percentage of BrdU+/DAPI+ cellscompared to total DAPI+ cells was measured. It was observed thatNefopam-treated human AF tumours contained significantly fewerproliferating cells as determined by BrdU incorporation (p<0.05, seeFIG. 4B).

Together, these results show that Nefopam preferentially inhibits thenumber of viable AF cells by reducing the rate of proliferation.

Example 5: Nefopam Decreases β-Catenin Levels in Primary HumanFibroblast Cell Cultures

Hyperplastic wounds are characterized by elevated β-catenin levelsduring the proliferative phase. The data described herein show thatNefopam has the capacity to modulate β-catenin levels particularly inmesenchymal-derived cells. To confirm that Nefopam can modulateβ-catenin levels in mesenchymal cells, immortalized human fibroblastcells were treated with Nefopam (see FIG. 5A). Nefopam treatmentresulted in an approximately 4-fold decrease in total β-catenin levelsin primary human fibroblast cell cultures compared to cultures treatedwith 0.1% DMSO as determined by densitometry analysis (p<0.05, see FIG.5B). Additional controls included in the experiments were Wnt3atreatment of cells (known to increase β-catenin expression) and effectsof Nefopam on cells treated with Wnt3a.

Example 6: Systemic Nefopam Decreases β-Catenin Levels and Wound Sizesin Tcf Mice

Next, to examine the effects of Nefopam on β-catenin levels during woundhealing, wounded tissue from Tcf mice were studied. Cutaneous woundswere generated using a biopsy punch procedure resulting in a 4 mmdiameter full thickness circular wound. Scale is in mm units. Westernblotting using an antibody against total β-catenin (see FIG. 6A)demonstrated a decrease in J3-catenin levels in cells cultured fromwounds derived from Tcf mice treated with Nefopam compared to thecontrol group 14 days post-wounding.

Furthermore, examination of the wounds upon autopsy showedNefopam-treated mice had scars significantly smaller in diametercompared to carrier (saline) treated controls at day 14 post-wounding(asterisk indicates a significant difference, p<0.001) (see FIG. 6B).

Example 7: Systemic Nefopam Reduces Hyperplastic Scar Size Induced byTGF-β

It is known that β-catenin protein levels increase during the earlystages of wound healing then fall through later stages relative tounwounded tissue. The normal rise and fall of β-catenin protein levelsare deregulated during hyperplastic wound healing where significantlyprolonged elevated levels of β-catenin are observed (see FIG. 7).

Following drug screen studies, effects of Nefopam were tested in vivousing mice. Both oral and intraperitoneal administration routes wereevaluated (40 mg/kg body weight, daily; 0.1% DMSO as control). In bothadministration routes, Nefopam was identified in the scrum as detectedusing HPLC (data not shown). 4 mm full thickness punch wounds were madein the skin, and Nefopam or control was administered daily afterwounding. To determine if Nefopam is effective in treating hyperplasticscars, a mouse hyperplastic scar model, in which TGF-β is injected priorto wounding resulting in a hyperplastic scar, was used. Importantly, thesame Nefopam treatment regimen, as described above, resulted in smallerscars as compared to control scars not treated with TGF-β (see FIG. 8).Thus, Nefopam is able to reduce scar size in both hyperplastic andnormal wound repair.

Example 8: Various Carriers can be Used for Topical Delivery of Nefopam

For skin wounds, an ideal product is a topical formulation of Nefopam.Topical Nefopam formulations using the following carriers were preparedand evaluated: carboxymethylcellulose (CMC), petrolatum, andhypromellose. The three carriers were tested in vivo to determine theformulation effective to deliver Nefopam through the skin. The resultsare illustrated in FIG. 9.

Example 9: Topical Nefopam Decreases Normal Scar Size in Mice

Cutaneous wounds were generated in Tcf mice using the biopsy punchprocedure described above. 6 mm diameter full thickness punch woundswere treated topically with either control cream of 1% Nefopam creamtwice daily for up to 21 days. It was observed that Nefopam treatmentresulted in a reduction in scar size by approximately one third comparedto controls. Table 1 indicates the average normal wound size (measuredin mm) in a mouse model at day 0 and following 21 days of daily topicaladministration of either 1% Nefopam cream formulated in petrolatumcarrier or carrier alone control cream. Averages are provided for 4wounds per group.

TABLE 1 Day 0 Day 21 Control Average size of wound (mm) 6 3.116666667 1%nefopam Average size of wound (mm) 6 2.02

4 mm punch wounds were generated in Tcf mice which were then treatedtopically with one of 1% Nefopam cream or control cream twice daily for14 days. The surface areas of scars formed after 14 days of treatmentwere measured using arbitrary units, where 100 arbitrary unitsrepresents the control cream treatment. Ten wounds were measured foreach treatment and data presented as mean and standard deviation. It wasobserved that scars in mice receiving Nefopam treatment weresignificantly smaller than those subjected to control treatment (p<0.05)where control treatment is 0% Nefopam (see FIG. 10).

Example 10: Various Excipients can be Used for Topical Delivery ofNefopam

According to certain embodiments of the invention the dosage form to beused for topical applications is BID 1% Nefopam HCl cream (oil in wateremulsion (O/W)). In these embodiments the application of the inventivecompounds may start approximately 5-7 days post-surgery and conclude atapproximately 30 days. In certain embodiments, application of theinventive compounds to the wound is by the patient. Therefore apreservative system is provided. Nefopam HCl is hydrophilic but thehydrochloride salt form has improved aqueous solubility and contains achiral centre and it is believed that there are two enantiomers in a50:50 ratio. The ideal pH for solutions of Nefopam HCl according to theinvention is 5.2 to 5.4. For skin wounds of humans, an ideal productmust be stable, spreadable, penetrate well, and ideally have low odor.Topical Nefopam formulations using a variety of combinations ofexcipients are disclosed herein.

Example 11: Topical Formulation with Transcutol® P and without CalciumGluconate

Table 2 below provides the ingredient concentrations (mg/g) of onetopical formulation of Nefopam HCl 1% cream according to the invention.

TABLE 2 Ingredients mg/g mg/g Nefopam HCl (as free base) 10.00 10.00Transcutol ® P 30.00 45.00 Cetyl Alcohol NF 40.00 40.00 Stearyl AlcoholNF 30.00 30.00 Liposorb ® S-20 (Polysorbate 60 NF) 40.00 40.00Propylparaben NF 0.50 0.50 Lexol (Isopropyl Myristate NF) 22.50 15.00Purified Water, USP 818.50 810.00 Methylparaben NF 1.00 1.00 CalciumGluconate NF — — EDTA 0.02 0.02 Purified Water, USP for 10% Solution4.50* 4.50* NaOH Sodium Hydroxide NF, qs to 5.0 to 5.6 0.50* 0.50* Total(mg) 1000.00 1000.00 *The quantity entered is the actual quantity addedto the batch to achieve an approximate pH of 5.3. pH of each batch wastaken at approximately 56° C. to 60° C.

General Processing Directions with Transcutol® and without CalciumGluconate:

-   -   1. Load cetyl alcohol, stearyl alcohol, polysorbate 60, and        isopropyl myristate into a suitable jacketed cream/ointment        processing kettle. Heat to 65° C. to 70° C. while mixing.    -   2. Load Propylparaben and mix to ensure complete dissolution.        Maintain the temperature at 65° C. to 70° C.    -   3. Load Transcutol® into a stainless steel container. Heat to        60° C. to 65° C. Add Nefopam HCl while stirring. Mix to form a        slurry. After approximately 10 minutes of stirring, add to Step        2 while mixing. The container will be rinsed with purified water        in commercial or GMP processing to affect a quantitative        transfer.    -   4. Add purified water into a heated, stainless steel tank,        equipped with a mixer. Stir while heating to 65 to 70° C. Add        EDTA and mix to dissolve. Add methylparaben and mix to dissolve.        Maintain the temperature at 65° C. to 70° C. Maintain the        temperature at 65° C. to 70° C.    -   5. Transfer step 3 into step 2. Qs to 99%. Homogenize and mix        for 30 minutes while maintaining the temperature at 65° C. to        70° C. while imparting a vacuum of −0.4 to −0.6 Atm.    -   6. Adjust the pH to 5.0 to 5.6 with 1N NaOH. Measured at 25° C.        Ensure complete homogeneity after the addition of the pH        adjusting agent by homogenizing and mixing for 5 minutes after        each addition. Qs with purified water if necessary.    -   7. Cool, while mixing and homogenizing, to 55° C. to 58° C. At        55° C., turn off the homogenizer. Maintain the vacuum while        slowly mixing and cooling to less than 30° C. Fill into 50 mL        glass jars, cap and label.

Example 12: Topical Formulation with Transcutol® and with CalciumGluconate

Table 3 below provides the ingredient concentrations (mg/g) of onetopical formulation of Nefopam HCl 1% cream according to the invention.

TABLE 3 Ingredients mg/g Nefopam HCl (as free base) 10.00 Transcutol ® P30.00 Cetyl Alcohol NF 40.00 Stearyl Alcohol NF 30.00 Liposorb ® S-20(Polysorbate 60 NF) 40.00 Propylparaben NF 0.50 Lexol (IsopropylMyristate NF) 22.50 Purified Water, USP 806.50 Methylparaben NF 1.00Calcium Gluconate NF 5.00 EDTA 0.02 Purified Water, USP for 10% 4.50*Solution NaOH Sodium Hydroxide NF, qs to 5.0 to 0.50* 5.6 Total (mg)1000.00 *The quantity entered is the actual quantity added to the batchto achieve an approximate pH of 5.3. pH of each batch was taken atapproximately 56° C. to 60° C.

General Processing Directions with Transcutol® and with CalciumGluconate:

-   -   1. Load cetyl alcohol, stearyl alcohol, polysorbate 60, and,        isopropyl myristate into a suitable jacketed cream/ointment        processing kettle. Heat to 65° C. to 70° C. while mixing.    -   2. Load propylparaben and mix to ensure complete dissolution.        Maintain the temperature at 65° C. to 70° C.    -   3. Load Transcutol® into a stainless steel container. Heat to        60° C. to 65° C. Add Nefopam HCl while stirring. Mix to form a        slurry. After approximately 10 minutes of stirring, add to Step        2 while mixing. The container will be rinsed with purified water        in commercial or GMP processing to affect a quantitative        transfer.    -   4. Add purified water into a heated, stainless steel tank,        equipped with a mixer. Stir while heating to 65° C. to 70° C.        Add methylparaben and mix to dissolve. Add calcium gluconate and        mix to dissolve. Maintain the temperature at 65° C. to 70° C.        Maintain the temperature at 65° C. to 70° C.    -   5. Transfer step 3 into step 2. Qs to 99%. Homogenize and mix        for 30 minutes while maintaining the temperature at 65° C. to        70° C. while imparting a vacuum of −0.4 to −0.6 Atm.    -   6. Adjust the pH to 5.0 to 5.6 with 10% NaOH. Measured at 55° C.        to 65° C. Ensure complete homogeneity after the addition of the        pH adjusting agent by homogenizing and mixing for 5 minutes        after each addition. Qs with purified water if necessary.    -   7. Cool, while mixing and homogenizing, to 55° C. At 55° C.,        turn off the homogenizer. Maintain the vacuum while slowly        mixing and cooling to less than 30° C. Fill into 50 mL glass        jars, cap and label.

Example 13: Topical Formulation without Transcutol® and without CalciumGluconate

Table 4 below provides the ingredient concentrations (mg/g) of onetopical formulation of Nefopam HCl 1% cream according to the invention.

TABLE 4 Ingredients mg/g Nefopam HCl (as free base) 10.00 Transcutol ® P— Cetyl Alcohol NF 40.00 Stearyl Alcohol NF 30.00 Liposorb ® S-20(Polysorbate 60 NF) 40.00 Propyl Paraben NF 0.50 Lexol (IsopropylMyristate NF) 22.50 Purified Water, USP 838.50 Methyl Paraben NF 1.00Calcium Gluconate NF — EDTA 0.02 Purified Water, USP for 10% 4.50*Solution NaOH Sodium Hydroxide NF, qs to 5.0 to 0.50* 5.6 Total (mg)1000.00 *The quantity entered is the actual quantity added to the batchto achieve an approximate pH of 5.3. pH of each batch was taken atapproximately 56° C. to 60° C.

General Processing Directions without Transcutol® and without CalciumGluconate:

-   -   1. Load cetyl alcohol, stearyl alcohol, polysorbate 60, and        isopropyl myristate into a suitable jacketed cream/ointment        processing kettle. Heat to 65° C. to 70° C. while mixing.    -   2. Load propylparaben into Step 1. Mix to ensure complete        dissolution. Maintain the temperature at 65° C. to 70° C.    -   3. Add 409.25 g of purified water into a heated, stainless steel        tank equipped with a mixer. Stir while heating to 65° C. to        70° C. Add EDTA and mix to dissolve. Add methylparaben and mix        to dissolve. Maintain the temperature at 65° C. to 70° C. Add        Nefopam HCl while stirring. Mix until dissolved. Maintain the        temperature at 65° C. to 70° C.    -   4. Transfer Step 3 into Step 2. Qs to 99%. Homogenize and mix        for 30 minutes while maintaining the temperature at 65 to        70^(° C.) while imparting a vacuum of −0.4 to −0.6 Atm.    -   5. Adjust the pH to 5.0 to 5.6 with 1N NaOH. Measured at 25° C.        Ensure complete homogeneity after the addition of the pH        adjusting agent by homogenizing and mixing for 5 minutes after        each addition. Qs with purified water if necessary.    -   6. Cool, while mixing and homogenizing, to 55° C. At 55° C.,        turn off the homogenizer. Maintain the vacuum while slowly        mixing and cooling to less than 30° C. Fill into 50 mL glass        jars, cap and label.

Example 14: Comparison of the Physical Aspects of the Cream Formulations

The table below compares the results of the physical tests of the four(4) formulae.

TABLE 5 Example 11 Example 11 with low with high Test Transcutol ® PTranscutol ® P Example 12 Example 13 Appear- White, shiny White, shinyWhite, shiny White, shiny ance smooth smooth smooth smooth cream. cream.cream. cream. Medium-low Medium Medium Medium viscosity** viscosity**viscosity** viscosity** Micro- Ultrafine, Ultrafine, Ultrafine,Ultrafine, scopic well well well well appear- dispersed disperseddispersed dispersed ance emulsion. No emulsion. No emulsion. Noemulsion. No (100X) evidence of evidence of evidence of evidence ofundissolved undissolved undissolved undissolved API. API. API. API. pH5.0- ≈5.3 ≈5.3 ≈5.3 ≈5.3 5.6*** **The viscosity determination issubjective and is based on moving a spatula through the cream todetermine resistance. ***The pH was taken with a pH meter, calibrated atpH 4.0 and 7.0. The temperature of the cream was ≈60° C. during thetesting. The pH meter was adjusted to 60° C. for the pH testing.

Example 15: Topical Treatment of Wounds with 1% Nefopam Cream in DurocPigs

The efficacy of a 1% Nefopam cream disclosed in Example 11 (30 mgTranscutol® P) applied daily to 2×2 cm excisional wounds and 2 cmincisional wounds when compared to cream vehicle alone (control). Thetopical formulation used in this Example was optimization to enhancestability and delivery of the Nefopam. The effects on wound repair andscar formation were quantified.

Four Duroc pigs (two males and two females) weighing 6-8 kg were usedfor this Example. Two 2×2 cm wounds were generated on the back of eachpig, one wound on each side. The wounds were generated by excising afull thickness section of skin (to the muscle layer). Half of the pigs(2 pigs, 4 wounds) were treated daily with cream vehicle (controls)while the other half (2 pigs, 4 wounds) were treated daily with 1%Nefopam cream. Meloxicam (analgesic; 5 mg/ml at 0.4 mg/kg IM) andPenicillin G (antibiotic; 300,000 UI/ml at 1 ml/20 kg IM) wereadministered to each pig after surgery. One week later, further surgerywas performed on these pigs to generate 2 cm incisional slit wounds (onewound on each side of the pig for a total of two incisional wounds perpig). These were generated using a scalpel, incising deep to the musclelayer. Both the 2×2 cm excisional wounds and the 2 cm incisional woundswere monitored and photographs taken each week to document healing. Thescar area was quantified using the AlphaEaseFC 4.0 program. All pigs(Nefopam treated and controls) were monitored for 4 weeks in the case ofpost-excisional wounding and 3 weeks in the case of post-incisionalwounding.

At four weeks post-excisional wounding, the pigs were sacrificed and thewound tissues were excised and fixed in 10% formalin for histologicalanalyses. Scar size was measured from the histological sections usingMasson-Trichrome stained sections cut across the wound perpendicular tothe skin. Serial sections were cut across the scar to identify thewidest diameter of each scar (mid aspect of the scar), and this sectionwas used to measure scar size. An observer blinded to the treatmentmeasured the scar size.

Table 6 below is a summary of histology measurements for 2×2 cmexcisional wounds on pigs treated with 1% Nefopam cream according toExample 11 vs. control at day 28 post-wounding; n=2 pigs/group.

TABLE 6 Control 1% Nefopam Cream Day 28 Average 52.7 34.9 StandardDeviation 10.8 3.9 95% Confidence Limit 4.5 1.0 T test to control N/A9.6E−08

Table 7 below is a summary of histology measurements for 2 cm incisionalwounds on pigs treated with 1% Nefopam cream according to Example 11 vs.control at day 21 post-wounding; n=2 pigs/group.

TABLE 7 Control 1% Nefopam Cream Day 21 Average 39.6 24.4 StandardDeviation 6.9 3.3 95% Confidence Limit 3.0 1.4 T test to control N/A1.3E−09

The excisional and incisional wound area and scar size data derived bothfrom the weekly photographic measurements of the scars and measurementsfrom histology sections, respectively, indicated that there was asignificant difference in scar size between Duroc pigs treated with 1%Nefopam cream and those treated with vehicle cream (control).Specifically, the surface scar images showed a markedly reduced scarsize through the duration of wound repair of the 2×2 cm excisionalwounds and the 2 cm incisional wounds over 28 days. Additionally, thescar size as determined from the final histological sections wassignificantly smaller at Day 28 for the 2×2 cm excisional wounds and atDay 21 for the 2 cm incisional wounds after Nefopam treatment comparedto control.

The results of these studies on wound repair and scar formation in theDuroc pig model subjected to excisional and incisional wounds andtreated with 1% Nefopam cream according to Example 11 or controlconfirmed the results from the earlier studies of wound repair and scarformation following Nefopam treatment (wild-type mouse model (seeExample 9) and pigs treated with initial cream formulations).

Specifically, the excisional and incisional wound studies in Duroc pigsdemonstrate that there is a significant improvement in wound repair andreduction in scar size formation following daily treatment with 1%Nefopam cream.

Example 16: Acne Treatment

According to certain embodiments of the invention, the cream dosage formis used to reduce scarring resulting from acne. In this example, 1%Nefopam HCl cream is applied twice daily directly to the wounded areafor 30 days or until the wound has disappeared.

Example 17: Treating a Human Wound with a Nefopam Cream

A patient sustained a cut from a saw approximately 1 cm in length on aknuckle. The wound was closed with non-dissolving sutures. On day 4after the injury, treatment with 1% Nefopam cream disclosed in Example11 (30 mg Transcutol® P) was initiated. The cream was applied twicedaily for 21 days. The sutures were removed after 9 days. The patientcontinued all of his regular daily activities. There were no adverseeffects from the treatment and the scar from the wound is virtuallyundetectable.

While this invention has been described with reference to illustrativeembodiments and examples, the description is not intended to beconstrued in a limiting sense. Thus, various modifications of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thisdescription. It is therefore contemplated that the appended claims willcover any such modifications or embodiments. Further, all of the claimsare hereby incorporated by reference into the description of thepreferred embodiments.

Any publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1-24. (canceled)
 25. A topical formulation of Nefopam comprising: (a) aneffective amount of a compound selected from Nefopam or a functionallyequivalent salt thereof; (b) a solvent/penetration enhancer; (c) atleast one fatty alcohol emulsifier; (d) an oil-in water emulsifier; (e)at least one preservative; and sodium hydroxide.
 26. The topicalformulation of claim 25, wherein the penetration enhancer is selectedfrom the group consisting of (a) diethylene glycol monoethyl and (b)isopropyl myristate, or both (a) and (b).
 27. The topical formulation ofclaim 25, wherein the at least one fatty alcohol emulsifier is selectedfrom the group consisting of (a) cetyl alcohol and (b) stearyl alcohol,or both (a) and (b).
 28. The topical formulation of claim 25, whereinthe oil-in-water emulsifier is polysorbate
 60. 29. The topicalformulation of claim 25, wherein the preservative is propylparaben. 30.The topical formulation of claim 25, wherein the Nefopam or functionallyequivalent salt thereof is present in an amount of about 10 m/g.
 31. Thetopical formulation of claim 26, wherein the formulation comprises thepenetration enhancer diethylene glycol monoethyl, present in an amountfrom about 30 mg/g to about 45 mg/g.
 32. The topical formulation ofclaim 26, wherein the formulation comprises the penetration enhancerisopropyl myristate, present in an amount from about 15 mg/g to about22.5 mg/g.
 33. The topical formulation of claim 27, wherein theformulation comprises the fatty acid emulsifier cetyl alcohol, presentin an amount of about 40 mg/g.
 34. The topical formulation of claim 27,wherein the formulation comprises the fatty acid emulsifier stearylalcohol, present in an amount of about 30 mg/g.
 35. The topicalformulation of claim 28, wherein the formulation comprises theoil-in-water emulsifier polysorbate 60, present in an amount of about 40mg/g.
 36. The topical formulation of claim 29, wherein the formulationcomprises the preservative propyl paraben, present in an amount of about0.5 mg/g.
 37. The topical formulation of claim 25, wherein theformulation further comprises an anti-fungal agent.
 38. The topicalformulation of claim 37, wherein the anti-fungal agent is methylparabenpresent in an amount of about 1.0 mg/g.
 39. The topical formulation ofclaim 25, wherein the formulation further comprises a chelating agent.40. The topical formulation of claim 39, wherein the chelating agent isEDTA present in an amount of about 0.2 mg/g.
 41. The topical formulationof claim 25, wherein the formulation further comprises a form ofcalcium.
 42. The topical formulation of claim 41, wherein the form ofcalcium is calcium gluconate present in an amount of about 5.0 mg/g. 43.A topical formulation of Nefopam comprising: (a) an effective amount ofa compound selected from Nefopam or a functionally equivalent saltthereof; (b) a penetration enhancer selected from (1) diethylene glycolmonoethyl in an amount from about 30 mg/g to about 45 mg/g; (2)isopropyl myristate in an amount from about 15 mg/g to about 22.5 mg/g;or both (1) and (2); (c) a fatty alcohol emulsifier selected from (1)cetyl alcohol in an amount of about 40 mg/g; (2) stearyl alcohol in anamount of about 30 mg/g; or both (1) and (2). (d) an oil-in wateremulsifier polysorbate 60 in an amount of about 40 mg/g. (e) apreservative propylparaben in an amount of about 0.5 mg/g. (f) achelating agent EDTA in an amount of 0.02 mg/g; and sodium hydroxide.44. The topical formulation of claim 43, wherein the sodium hydroxide isadded in an amount sufficient to reach a pH of about 5.0 to about 5.6.